The objective is to identify general characteristics of antigens that enable or enhance generation of MHC class I associated epitopes. Listeria monocytogenes (LM), a bacterium that infects and secretes proteins into the cytosol of cells, will be used to deliver antigens. This system has the following advantages: 1) the amount of antigen delivered can be determined; 2) antigen delivery can be terminated rapidly with antibiotics; and 3) antigens can be metabolically labeled in the host cell. By quantifying epitopes extracted from MHC molecules, the investigator has determined that 30-40 molecules of p60, a secreted LM protein, are degraded to make one p60 217-225 CTL epitope. The first goal is to determine which regions of p60 influence the efficiency of its processing into p60 217-225. It will be determined if amino acid residues within or flanking the epitope are critical for its generation. Degradation signals within p60 and their influence on antigen processing will be determined. The second goal is to determine if findings with p60 can be generalized. The requirements for processing of LM-secreted ovalbumin and influenza virus nuclear protein into MHC class I associated epitopes will be investigated. The third goal is to investigate the role of different protein degradation signals on epitope generation. The role of PEST sequences and the N-end rule on antigen stability and epitope generation will be investigated. The investigator will also construct antigens that contain degradation signals which enhance ubiquitination and cell cycle-dependent protein degradation, and determine if these signals enable entry into the MHC class I antigen processing pathway. These studies will determine requirements for efficient antigen processing. Knowing what determines the efficiency of MHC class I antigen processing is essential for the development of vaccines that elicit CTL.